Nuclear Import/Export Flashcards
NLS, 2 types
Classic NLS: most common, consists of short stretch of positive amino acids
Bipartite NLS: composed of 2 short stretches of basic amino acids and a 7-10 long spacer sequence
NLS definition
nuclear localization signal: an amino acid sequence both necessary and sufficient for cytosol-to-nuclear targeting
necessary: if mutated protein can’t target the nucleus then it gets lost in the cytosol
sufficient: if it was linked to a non-nuclear protein then fusion protein not capable of getting to the nucleus
NES, Definition
nuclear export signal: LQCLMSL, common NES, L is hydrophobic. Exportin binds to NES to lead to export, RanGTP required to assemble export complex (Exporting-RanGTP-protein) which is sent to cytoplasm and RanGTP hydrolysis releases cargo
Major Players in Nuclear Import/Export
NPC: cytoplasmic filaments, central channel and nuclear basket
Cargo: any protein that has an intrinsic, genetically encoded NLS
Importin alpha: protein that recognizes NLS
Importin beta: binds importin alpha and the cytoplasmic filaments of the NPC
RanGTP: active G protein
RanGDP: inactive G protein
RanGAP1: GTPase activating protein, GTPase hydrolyzes GTP to GDP
RCC1: guanine exchange factor (GEF) that exchanges GDP for GTP
Exportin: protein that recognizes NES
Step 1 in Nuclear Import/Export
Importin alpha binds the NLS in the cargo protein and importin beta binds to importin alpha
Step 2
importin alpha/beta/cargo-NLS complex associates with the cytoplasmic filaments of the NPC via microtubules
Step 3
complex is pushed through the pore and associates with the nuclear basket
step 4
RanGTP binds to Importin
step 5
causes conformation change of importin beta and this leads to the entire complex to dissociate from each other and the nuclear basket
step 6
importin beta/RanGTP complex goes down the GTP concentration gradient via the NPC because of the high concentration of GTP in nucleus
step 7
RanGAP1 senses the RanGTP in the cytoplasm and associates with it, hydrolzes GTP to GDP creating RanGDP which causes Importin B to dissociate from RanGDP
step 8
RanGDP heads down its concentration gradient via NPC to the nucleus
step 9
RCC1 (a gef) associates with GDP and exchanges it from GDP to GTP, RanGTP is now free to undergo another round of export
Export of Importin alpha step 1
importin alpha has an NES that is exposed when released from Importin beta and its cargo
Export of Importin alpha step 2
exportin senses the NES and binds to it, and then associates with RanGTP
Export of Importin alpha step 3
the RanGTP/Exportin/Importin alpha-NES complex flows down the RanGTP concentration gradient through the NPC to the cytoplasm because there is a lot of RanGTP in the nucleus
Export of Importin alpha step 4
RanGAP1 senses the GTP bound to exporting and converts it to RanGDP
Export of Importin alpha step 5
causes Importin alpha to dissociate from Exportin and RanGDP to dissociate from Exportin
Export of Importin alpha step 6
RanGDP flows down ranGTP concentration gradient through NPC to nucleus, RCC1 senses RanGDP converts it to RanGTP and Exporting has an NLS causing it to become a cargo protein targeted for import
Can proteins without an NLS be imported?
yes, they piggyback nuclear protein import… a nascent protein lacking an NLS binds to an NLS-containing protein in the cytosol and targeting and import of the protein-protein complex into nucleus is mediated by importing receptors as usual
two mechanisms used to maintain mb asymmetry
1) lipid composition
2) modification and orientation of integral mb proteins (IMPs), lumenal domain of IMP (in lumen and forms extracellular domain on face of PM) and cytoplasmic domain (always in cytoplasm)
3 steps of cotranslational translocation of soluble protein
1) translation of mRNA on free ribosome in cytosol, the N terminus of nascent polypeptide emerges from ribosome containing a signal sequence which is recognized by an SRP and the SRP binds to ribosome to stop translation
2) SRP targets entire complex to the surface of the ER and the SRP binds to an SRP receptor, the SRP is released from GTP hydrolysis that resulted in conformational change and the ribosome binds to the cytosolic side of the translocon, this binding results in continuation of protein translation, the signal sequence interacts with interior of translocon and causes a conformational change that opens the pore ring and displaces the ‘plug’
3) Growing protein moves through the translocon (translocated across ER mb.) and just before the signal sequence enters the lumen it is cleaved by the signal peptidase and as the nascent protein enters the lumen its glycosylated and gets folded by reticuloplasms, following termination of translation and translocation the ribosome is released from the translocon and returns to cytosol for another round of protein import, this release causes closing of translocon pore and return of ‘plug’
4 steps of cotranslational insertion of an integral mb protein into the RER
1) N terminus of nascent polypeptide enters translocon, first or only TMD enters interior of translocon and serves as a stop-transfer sequence (stops any further translocation of nascent protein through translocon), orientation of mb protein must be N-lumen, C-cytosol
2) Signals translocon to open laterally and TMD segment is released into mb lipid bilayer, positive-outside rule applies here where TMD is re-oriented by translocon so +vely charged residues face the cytosol and are released into mb like that (+vely charged AA’s determine topology of mb proteins)
3) Synthesis of the proteins cytosolic-facing C terminus residues, final mb orientation: N-lumen, C-cytosol
4) Synthesis of the proteins C terminus residues, final mb orientation: N-cytosol, C-lumen
four general steps of vesicle transport:
1) Budding: ‘cargo’ containing vesicle buds off the donor mb. compartment
2) Transport: nascent vesicle is transported through the cytosol to the recipient mb. compartment
3) Fusion: vesicle ‘fuses’ with the proper recipient membrane compartment, vesicle mb and cargo proteins are incorporated into recipient compartment
4) Retrograde Transport: entire process of budding and fusion is repeated and can occur in reverse direction
four transport pathways
1) biosynthetic: materials transported from ER to Golgi, to Endosomes, then to Lysosomes/Vacuoles or to Plasma Mb
2) constitutive: ER derived materials transported from Golgi to PM, secretory transport vesicle mb components are brought into PM and ‘cargo’ is released into extracellular space
3) regulated: occurs only in specialized cells, ER derived materials from Golgi are stored in secretary granules and in response to cellular signal secretary granules fuse with PM and release cargo into extracellular space
4) Endocytic: operates in opposite direction of secretory pathway, materials from PM and/or extracellular space incorporated into cell then transported to Endosomes and to Lysosomes/Vacuoles
Explain the structure of the ER
highly complex network of mb-enclosed rod-like tubules and sheet-like cisternae, has multiple subdomains which are distinct regions of the ER network that possess unique morphologies and/or functions
Define the subdomains of the ER
RER: mostly cisternae with bound ribosomes involves in protein and mb. phospholipid synthesis
SER: mostly curved tubules lacking ribosomes, involves in Ca2+ storage and hormone synthesis
outer nuclear mb: continuous with RER and contains attached ribosome
MAM and PAM: regions of ER that make direct contact with mitochondria or PM and are involved in mb. lipid exchange
ERES: regions where transport vesicles bud off from the ER en route to golgi
steps of N-linked protein glycosylation
i) core glycosylation: 13-step process, various ER mb-bound glycosyltransferases synthesize the core oligosaccharide, each enzyme adds a specific sugar to a specific position on the growing core one at a time, final step involves linking core oligosaccharide to specific N residue on a soluble or integral mb. protein that is still being synthesized via co-translational translocation pathway
ii) core modification: after transfer to nascent protein, 12-sugar core oligosaccharide is gradually trimmed and modified - first two terminal glucose units are removed by glucosidase I & II, the nascent glycoprotein is properly folded by recticuloplasmins (BiP), also subjected to ER quality control (ensures protein possesses correct sugar attachments)
explain ER protein quality control during N-linked glycosylation
nascent glycoprotein binds to calnexin (folding protein), glucosidase II removes last glucose unit, protein is released from calnexin and is properly folded to function in the ER or transported from ER to Golgi then to somewhere in Endo mb. system
What happens if a protein is misfolded in the ER?
1) it is recognized by GT monitoring enzyme, which adds back a single glucose residue to terminal end of trimmed core and misfiled protein binds to calnexin again and entire process continues until protein is properly folded
2) destroyed via ERAD pathway
3) UPR pathways - accumulation of misfolded proteins cause ER stress that signal these pathways to relieve stress
Explain ERAD pathway
1) misfolded protein translocated back out of ER into cytosol via translocon (retro-translocation)
2) in cytosol: oligosaccharide chains are removed and poly-ubiquinated (linked to a chain of repeating ubiquitin units), serves as a signal for ER protein degradation via degradation by the proteasome - Ub protein binds to lid of proteasome, Ub chain is removed and recycled and the protein is threaded into proteasome where its degraded (via proteolysis) and amino products are reused for protein new synthesis
Explain the 3 UPR pathways
In ER stress conditions, UPR pathway activated and BiP is released
1) IRE1-mediated UPR pathway: BiP released from IRE1 in order to aid in the folding of accumulating misfolded proteins, IRE1 is activated and splices XBP1 which increases the levels of ERAD related proteins
2) PERK-mediated: BiP released from PERK, PERK sensors dimerize and become active and phosphorylate, inhibiting the protein translation factor eIF2alpha which decreases protein synthesis in the cell and the molecule chaperones can focus on the accumulated misfiled proteins and alleviate stress
3) ATF6-mediated: BiP released from ATF6, ATF6 moves from ER to golgi and the transcription factor domain of ATF6 is cleaved off and targets the nucleus which up-regulates a number of genes encoding molecular chaperones, export components and ERAD components to alleviate ER stress
two main functions of COP vesicles
1) recognize and concentrate specific protein (& lipid) components that will be incorporated into the budding vesicle (e.g. cargo destined for recipient compartment and the molecular machinery required)
2) mediate ERES membrane curvature and the formation of the budding vesicle
three major classes of coat proteins
1) COPII: move forwards (anterograde transport) from the ERES to the Golgi
2) COPI: move backwards (retrograde transport) from the Golgi to the ER and ‘backwards’ within the golgi
3) Clathrin: move from the Golgi to the PM
5 steps of transport vesicle assembly
1) COPII component Sar1-GDP is recruited from cytosolic surface of ERES and binds to GEF which generates Sar1-GTP
2) Sar1-GTP integrates into outer leaflet at ERES and causes mb. curvature of ERES membrane
3) Sar1-GTP recruited other COPII components Sec23 and Sec24 which promote further bending of ERES, Sec24 binds to cytosolic-facing domains of selected integral mb proteins
4) additional COPII components - Sec13 and Sec31 - join the growing coated vesicle bud and act as an outer scaffolding of the coat
5) after coat assembly, the vesicle bud pinches off from the ERES and is trafficked to the proper recipient mb (golgi) and before fusion the COPII coat disassembles (Sar1GTP converted back to Sar1GDP and released to cytosol)
4 steps of vesicle fusion
1) Recognition of incoming vesicle and recipient mbs, this is mediated by Rab proteins (requires GTP)
2) Tethering of incoming vesicle to recipient mb: complementary mb-bound GTP-Rabs recruit various recipient factors like tethering proteins which interact to form a molecular bridge and mediate vesicle membrane-recipient membrane contact by bringing two membranes close together
3) Docking of vesicle at recipient membranes: mediates by SNARE proteins (unique snares associated with diff. membranes - show specificity), v-SNARES and t-SNARES interact to form a SNARE complex that pulls the vesicle and recipient membranes closer together
4) Fusion of vesicle and acceptor membrane: SNARE complex fuses membrane and vesicle which results in lateral movement of vesicle membrane proteins (cargo and receptor proteins) into recipient membrane and cargo proteins released into lumen of acceptor organelle
Difference between vSNARE and tSNARE proteins
vSNARES: found on transport vesicle membranes incorporated into the vesicle membrane at the site of budding on the donor compartment
tSNARES: found on target ‘acceptor’ membranes
Explain retrograde transport of ‘escaped’ resident soluble ER proteins
- *returned from CGN back to ER by specific ER retrieval signals, CGN-to-ER**
- soluble ER proteins possess a KDEL sequence (ER retrieval signal) and ‘escaped’ ER proteins are recognized in the CGN lumen by the KDEL receptor
- COP I mediates the formation of transport vesicles at the CGN and return the soluble ER protein-KDEL receptor complex back to the ER
- at the ER, KDEL releases the resident ER protein and returns to the CGN via COPII vesicles
list and describe sub compartments of the golgi complex
CIN : cis golgi network, consists of an interconnected network of tubules and vesicles adjacent to the ERES, serves as a sorting station: its the destination of COPII vesicles and site of COPI vesicle assembly for retrograde transport or anterograde transport as CGN matures into golgi complex
Golgi Cisternae: series of three or more large flattened cisternae that make up majority of the organelle, divided into cis, medial and trans cistern and these are the sites where golgi metabolism occurs (synthesis of complex polysaccharides)
TGN: trans golgi network, interconnected network of tubules and vesicles, serves as a sorting station: involves in anterograde transport of materials from golgi to places in endomb., site of clathrin vesicle assembly, site of secretory vesicle/granule assembly for PM transport, site of COPI vesicle assembly for retrograde transport
function of golgi
processing plant, synthesizes complex polysaccharides, modification of proteins and lipids, transport and sorts proteins (from ER to gold to PM - anterograde, from PM to golgi to ER - retrograde, sorting occurs at CGN or TGN) glycosylation (cis, medial and trans golgi cisternae possess unique glycosyltransferase enzymes, N-linked glycosylation completed in golgi complex)
explain the cisternal progression/maturation model of movement of materials through the golgi complex
golgi sub compartments are considered dynamic structures and each sub compartment continually moves from the cis to the trans side of the golgi complex. COPII-coated transport vesicles from the ER carrying new ‘cargo’ proteins arrive at the cis face of the golgi complex, and incoming vesicles fuse together to form a new CGN, the newly formed CGN moves forward into the golgi stack and matures into the cis cisternae (anterograde), each cisternae matures into the next in the complex (chemical composition constantly changing), COPI-coated vesicles transport resident golgi enzymes retrograde through the complex, TGN eventually disperses into various types of vesicles; each which deliver certain ‘cargo’ from the TGN to different compartments in endomb.
what do clathrin coated vesicles do in movement of materials through golgi complex?
transport cargo to endosomes or lysosomes, including M6P-tagged cargo proteins destined for lysosome
what do secretory vesicles do in movement of materials through golgi complex?
transport cargo to the PM and extracellular space
what do secretory granules do in movement of materials through golgi complex?
eventually fuse with the PM and release their ‘cargo’ in the extracellular space
what is the fate of newly-delivered cargo at the CGN
- soluble and mb ‘cargo’ proteins from the ER either remain in the golgi or move to other compartments in the endo mb. system
- most ER resident proteins are retained in the ER by being excluded from budding COPII-coated transport vesicles at the ERES
Explain retrograde transport of ‘escaped’ resident ER membrane proteins
- most resident ER mb. proteins possess a cytosolic-facing, C-terminal KKxx sequence
- KKxx sequence on ‘escaped’ ER mb proteins at CGN is recognized by COPI
- COPI-coated vesicles return resident ER membrane proteins (& the KDEL receptor with its soluble protein ‘cargo’) back to ER through retrograde transport
Significance of Golgi’s association with the cytoskeleton
-GRASPS and Golgins
golgi matrix proteins link the golgi complex to the cytoskeleton, positioning and movement of the golgi complex within the cell is controlled by its interaction with the cytoskeleton
- Golgi ReAssembly and Stacking Proteins: serve as tethering proteins to link different golgi sub compartments together, assist in organization of golgi complex
- *GRASP55/65 results in disassembly of golgi complex**
- Golgins are long filamentous proteins that tether various parts of golgi to the cytoskeleton and other sub cellular structures, they dimerize and are regulated by G-proteins that anchor them to the golgi
Example of how modifications of proteins in golgi leads to protein targeting
- *targeting sequence to lysosome**
1) lysosomal protein arrives in CGN from RER
2) in cis golgi cisternae: enzyme recognizes a signal patch in the protein, N-acetylglucosamine-1-phosphate is transferred to a specific mannose residue from a nucleotide sugar donor
3) in medial golgi: N-acetylglucosamine group removed by a glycosidase and lysosomal enzyme now contains mannose-6-phosphate group
4) recognized by Mannose-6-Phosphate receptors (MPRs) in TGN, enzyme is targeted/sorted to lysosome
Explain protein glycosylation in golgi complex
glycosylation to the core oligosaccharide in the golgi complex serve as a targeting signal for proteins destined for the lysosome:
- in the cis cisternae: 2 mannose sugars in the core oligosaccharide are phosphorylated (N-linked oligosaccharides synthesis begins in ER, O-linked synthesis occurs in golgi)
- in the TGN: ‘cargo’ proteins with M6P groups are packaged into lysosomal-destined transport vesicles
- ‘cargo’ proteins without a M6P are packaged into other TGN transport vesicles/granules destined for PM (via secretory pathway)
lysosome function
digestive organelle, degrades all types of macromolecules and cellular organelles/components (autophagy), degradation products transported to cytosol, low pH in lysosome lumen maintained by mb-bound ATPase proton pump
Steps of clatherin vesicle formation and trafficking soluble proteins to the lysosome
- biosynthetic pathway*
1) Arf1-GTP binds to the TGN mb. which results in initial curvature of mb
2) GGA adaptor proteins are recruited from cytosol to TGN surface, act as linker proteins between mb. and clatherin
3) clatherin binding promotes further curvature of the mb. (driving force is the hexagon to pentagon transition in the triskelions)
4) dyamin polymerizes around the stalk of the budding vesicle, undergoes hydrolysis which causes a conformational change and release of the vesicle from the mb (pinching off)
5) after vesicle release the clatherin coat disassembles (Arf1-GTP to Arf1-GDP) and is recycled back to cytosol
6) Nascent vesicle fuses with late endosome (mediated via tabs, v-/t-snares)
7) M6P receptor dissociates from its cargo protein complex due to the pH-receptors recycled back to TGN
8) lysosomal proteins partitioned into a specific place in the endosome that fragments from rest of the organelle - MVB
9) MVB fuses with lysosome and contents are released - lysosomal enzymes are activated (low pH) and any other material (from PM) is degraded
Define the two main processes for internalization
1) endocytosis: selective internalization of PM components, two types: bulk-phase endocytosis and receptor mediated endocytosis
2) phagocytosis: uptake of large, particulate materials from the extracellular space of specialized cells (cellular eating)
what is bulk-phase endocytosis
‘pinocytosis’ or ‘cellular drinking’, responsible for non-specific uptake of extracellular fluids and plasma mb. protein turnover (PM recycles every 20-90 mins)
explain steps of receptor-mediated endocytosis
1) PM transmb. receptor becomes activated by binding to specific, soluble extracellular ligand, receptor-ligand complex diffuses laterally in the PM and accumulates in coated pits (inner leaflet of PM at coated pits enriched in unique mb. phospholipids that serve as a signal for recruiting adaptor protein AP2)
2) AP2 adaptors at the cytosolic-face of the coated pit form the inner layer of the ‘coat’ and recruit clathrin triskelions from cytosol to self-assemble and form the outer scaffolding of the coat on growing vesicle bud (clathrin polygon is driving force for mb. curvature)
3) clathrin-coated vesicle pinches off from PM via dynamin, clathrin coat disassembled,nascent endocytic vesicle with transmb. receptor-soluble ligand ‘cargo’ complexes targets to and fuses with early endosome (vesicle transport and fusion mediated by cytoskeleton and tabs, v/t-SNARE complexes)
4) acidic interior of early endosome causes dissociation of transmb. receptor from its soluble ligands and they’re sorted to different portions of the early endosome (recycling and sorting compartment)
5) recycled compartment detaches and traffics back to PM, sorting compartment contains ‘cargo’ and any PM receptors destined for degradation in lysosome
6) sorting compartment matures into a late endosome, endocytic ‘cargo’ proteins’ concentrated into late endosome fragment aka MVB
7) MVB fuses with lysosome, all MVB materials are released into lumen of lysosome, soluble ‘cargo’ proteins from the TGN are activated due to acidic pH, soluble endocytic ‘cargo’ are degraded by lysosomal acid hydrolyses and intralumenal vesicles with PM-deried receptors are degraded
explain the endosymbiotic theory of the mitochondria
explains how large host cell ingested other cells (bacterial cells) and developed symbiotic relationships with one another
mitochondria morphology
double mb. bound organelle: outer mb is permeable to ions and small molecules, it contains porins (barrel shaped integral mb. proteins w a large internal channel), inner mb. lies adjacent to outer and forms folds (cristae) that extend into organelles interior
- it is impermeable, the intermb. space is high in H+ and is the site of ATP synthase
- matrix is the aqueous interior that contains mitochondrial genome and ribosomes
- tubules are mobile and can fuse with one another or split apart (fission), mitochondrial fission occurs at the end of G1 and during apoptosis
- defects in mitochondrial network correlate with progression of numerous neurodegenerative diseases (alzhiemers, parkinson)
steps of mitochondrial matrix protein targeting
1) in cytosol: nascent protein recognized by cytosolic chaperones that maintain the protein in a partially unfolded, import-competent state
2) at surface of mitochondrion: matrix protein’s N-terminal pre sequence is recognized by TOM complex that forms a channel allowing for protein translocation across inner mb., TIM23 complex is connected to TOM allowing for translocation across intermb. space as well (driven by electrochemical potential, positive presequence attracted to less positively charged matrix)
3) as matrix’s protein’s N terminal pre sequence exits TIM23 channel, binds to matrix-localized mitochondrial chaperone (mtHsp70) which acts as a molecular motor (undergoes ATP-dependent conformational changes that pulls protein into matrix and prevents back sliding)
4) N-terminal pre sequence cleaved by matrix-localized protease (MPP) and mtHsp70 and other co-chaperones involved in proper folding of cleaved mature protein in matrix
define TOM, TIM23, TIM22 complexes
TOM: translocase of the mitochondrial outer mb., contains a number of integral mb. proteins: receptor (binds to positively charged pre sequence), channel (barrel-shaped, mediates protein translocation), accessory proteins (serve as scaffold to mediate protein transfer from receptor to channel)
TIM23: translocate of mitochondrial inner mb. domains of Tim23 interact with TOM domains
TIM22: responsible for insertion and assembly of inner membrane proteins, proteins passed from TOM to TIM22 are transferred laterally into lipid bilayer
Mitochondria and Parkinson’s Disease
healthy mitochondria import PINK1 into the intermb. space via TOM, PINK1 is cleaved by MPP and the remainder remains associated with inner mb. and is degraded by PARL and a protease
damaged mitochondria are depolarized and PINK1 can’t pass through TOM and TIM so it accumulates on the outer membrane and recruits Parkin, Parkin stimulates the poly-ubiquitination of outer mb. proteins and signals autophagy, only neutrons die because the production of dopamine requires a ton of energy given by the mitochondria and are sensitive to the loss of the energy source
chloroplast function
semi-autonomous plant cell organelle, site of photosynthesis
- Co2 and H20 + sunlight lead to O2 and sugars which goes through aerobic respiration in mitochondria and produce CO2 and ATP
- highly mobile in cells and move along cytoskeleton elements via molecular motors
chloroplast morphology
- envelope: consists of outer and inner mbs.
- outer mb: contains porins, not as permeable to small molecules as mitochondria
- intermb space
- inner mb: highly impermeable, contains transporters
what are thylakoids, thylakoid mbs and thylakoid lumen
thylakoids: internal mb. system, flattened membranous discs arranged in stacks (grana) or between stacks (stroma)
thylakoid mbs: site of ATP synthase, maintain H+ gradient in thylakoid lumen
thylakoid lumen: aqueous interior of thylakoid, high H+ conc., equivalent to intermb space in mitochondria
define stroma
between thylakoid stacks and inside envelopes, aqueous interior, contain enzymes involved in carbohydrate synthesis, contain plastid genome, contains ribosomes (used for translation of plastid genome-encoded proteins), encodes ribosomal proteins
define stromules
they connect chloroplasts, they’re long stoma-filled tubules that are highly dynamic and can rapidly extend and contract allowing for metabolite transfer, communication etc. between chloroplasts and other organelles
chloroplast protein targeting steps
1) stromal-destined proteins possess N-terminal targeting signal known as transit peptide which is recognized by the TOC complex
2) transit peptide binds to receptor protein of TOC complex (Toc34) in a GTP-dependent manner, partially unfolded stroll protein moves through the TOC complex and then through the TIC complex
3) As it exits the TIC complex, stromal chaperone protein Hsp93 serves as molecular motor that drives translocation across the envelope (ATP-dependent conformational changes that pull protein into stroma and prevent back sliding)
4) transit peptide cleaved by stroll processing enzyme, soluble co-chaperone Hsp60 in stroma ensures the cleaved, mature protein is properly folded
5) TOC and TIC complexes involved in insertion and assembly/import of other chloroplast proteins: outer&inner envelope mb. proteins: inserted into proper mb. by being transferred laterally from TOC or TIC, thylakoid proteins: nuclear encoded and targeted from cytosol in 2 steps: 1. imported into stroma (removal of transit peptide exposes thylakoid transfer domain) 2. thylakoid transfer domain mediates protein import into lumen or into mb
peroxisomes function and morphology
responsible for: lipid metabolism, oxidation of fatty acids and AA’s, neutralization of free radicals through process that produces large amounts of H2O2 which is converted to water and oxygen
- has crystallized core that is the location of urate oxidase: enzyme that catalyzes reaction of prix acid to allantoin in mammals
what is biogenesis in peroxisomes
- group I PMPs are post-translationally inserted either directly into the pER subdomain or first into the general ER then routed to the pER, all matrix proteins and group II PMPs are sorted post-translationally from cytosol to daughter peroxisomes
protein targeting in peroxisomes
1) cytosolic PEX5 and PEX7 recognize their cargo proteins via binding of specific targeting sequences PTS1 and PTS2, carl-loaded PEX5 associates with the mb. via interactions with PEX13 and PEX14
2) PTS1 and PTS2 bound cargo is released to the matrix, and the receptors are recycled back into the cytosol via a machoism that requires ATP-dependent ubiquitination of PEX5 by PEX4 and the RING complex comprised of PEX2, PEX10 and PEX12
3) ubiquitinated PEX5 is removed from mb. via action of the ATPases PEX6 and PEX1 which are tethered by APEM9
how does chloroplast and mitochondria protein targeting differ
there is 6 possible protein targeting areas in chloroplasts vs 4 in mitochondria, there is a transit peptide on the N-terminal of the nascent protein vs an A.A. pre sequence, TOC and TIC for chloroplasts vs TOM and TIM, TOC and TIC are not mediated by electrochemical potential though, the chaperone protein is Hsp93 in chloroplasts and mtHsp70 in mitochondria
